HIV-1, the causative agent of acquired immunodeficiency syndrome (AIDS), requires reverse transcriptase (RT) to copy its single stranded RNA genome into a double stranded DNA copy for integration into the host cell genome. Although almost all aspects of HIV-1's life cycle have been targeted (1-3), many of the drugs that have been effective in clinically are nucleoside reverse transcriptase inhibitors (NRTIs). However, treatment with NRTIs is limited by their toxicity to the host (presumably through their interaction with human mitochondrial DNA polymerase γ (4, 5) and the ability of the virus to mutate and acquire resistance (6). Other factors that effect the ability of these inhibitors to reduce viral replication are uptake, transport, metabolism, and incorporation of the drug. All clinically used nucleoside analogs lack 3′ hydroxyl groups and are metabolically activated by host cellular kinases to their triphosphate forms. These agents include 3′-azido-3′-deoxythymidine (AZT or Zidovudine), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T or Stavudine), β-L-(−)-2′,3′-dideoxy-3′-thiacytidine (3TC or Lamivudine), 2′,3′ dideoxycytodine (ddC or Zalcitabine) and 2′,3′ dideoxyinosine (ddI or Didanosine).
The structures of the FDA-approved drugs, abacavir (1592U89) and d4T, are unique because they contain a 2′,3′-unsaturated bond in the deoxyribose ring. Unlike d4T, abacavir contains a novel carbocyclic ring instead of the sugar ring (FIG. 1). Abacavir has been shown to be a potent and selective inhibitor of HIV-1 replication (7). The metabolic activation of this analog is unique. It is phosphorylated by adenosine phosphotransferase to a monophosphate and further metabolized in several steps to the triphosphate dGTP analog (−) carbovir-TP (CBV-TP)(8, 9). CBV-TP is thought to be the agent responsible for antiviral activity (8, 10-12). Abacavir has a promising pharmacokinetic profile, owing in part to its modified amino group at the 6 position of the purine ring (7, 13). Viral resistance to abacavir develops relatively slowly and cross-resistance between it and other nucleoside analogs is minimal (7, 14, 15). When screened against human DNA polymerases α, β, and γ, CBV-TP was found to be more selective for HIV-1 RT than AZT or dideoxynucleoside triphosphates (16, 17).
Although the resistance profile for abacavir is very good, studies in cell culture and clinical trials have isolated viral mutants in response to prolonged passage or treatment with the drug (14, 18, 19). Three mutations in HIV-1 RT have been found to be necessary to confer as high as an 11-fold resistance: methionine 184 to valine (M184V), leucine 74 to valine (L74V), and lysine 65 to arginine (K65R) or tyrosine 115 to phenylalanine (Y115F). The first mutation isolated in response to abacavir is the Ml 84V mutation (RTM184V) and has been associated with a 2 to 4 fold reduction in the virus's susceptibility. HIV-1 with this mutation has shown a 500-1000-fold resistance in the clinic to 3TC (20, 21). Mechanistic studies have shown that RTM184V is 30 to 140 fold more selective than wild type RT (RTWT) in distinguishing between dCTP and 3TC-TP depending on the primer template used (22) showing a good correlation between in vitro results and clinical findings.
While a steady-state kinetic analysis of deoxynucleoside triphosphate (dNTP) analogs has given a necessary initial inhibitory evaluation, (16, 17, 23) this approach cannot elucidate the detailed interaction of the drug with RT at the polymerase active site. The reason for the limited scope of this type of analysis is the inability to resolve kinetic steps masked by the rate-limiting step of a reaction. This point is of particular importance with the reaction mechanism of RT. RT follows an ordered reaction pathway (24). The first step involves binding of the DNA or RNA substrate to the enzyme to form an E•DNA complex with a dissociation constant (Kd) in the nanomolar range. This step is followed by the binding of a deoxy-nucleoside triphosphate (dNTP) to form the ternary complex (E•DNA•dNTP). The binding of dNTP is a two step process with an initial loose complex followed by a tighter binding complex as the enzyme undergoes a rate limiting conformational change for catalysis (kpol) and checks base geometry and pairing. Once the conformational change has taken place and the reactants are properly aligned at the active site for catalysis, the 3′ hydroxyl of the elongating strand attacks the a phosphate of the dNTP in a rapid chemical step. The rate-limiting step for the overall reaction (kss) is the release of the elongated DNA from RT. This is the step being analyzed during steady-state kinetic analysis.
The nucleoside compound 9-(2,3-Dideoxy-β-D-glycero-pent-2-enofuranosyl)guanine (d4G) previously has been reported to be inactive against HIV, whereas the carbocyclic analog carbovir has exhibited significant activity. (25, 10) More recent studies suggest that D4G may be unstable under acidic conditions and can retain anti-viral activity if buffered to neural or basic pH. (33) 
The optimal nucleoside combination therapy should include compounds that i. Interact synergistically (i.e., potentiate anti-HIV activity when combined); ii. Have different resistance profiles; and iii. Are active in all HIV infected cell types. The abacavir/3TC/AZT combination used presently falls short of these goals because of abacavir's lack of synergy and overlapping resistance profile with 3TC.
One area where the abacavir/3TC combination's weaknesses are most apparent is in resting cells. Resting cells are known as important reservoirs for HIV infection.26 In resting infected cells, deoxy-thymidine analogs have been shown to be relatively ineffective.27 This dampens AZT's effect in resting cells leaving abacavir and 3TC as the most active agents. The additive interaction between abacavir and 3TC would be sub-optimal under these conditions, however. The abacavir/3TC combinations could also serve to increase subpopulations of MI84V virus that both compounds select for.28-32 The presence of M184V would decrease the activity of 3TC by around 500-fold30, 32 leaving abacavir to fight the HIV infection alone with a 2-4 fold decrease in its activity due to the M184V mutation.31 This may explain the relative ineffectiveness of the drug combination in certain circumstances.